Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, UK (E.E.-K., Z.M.A.-M., A.R.-H., J.B., B.A.); Clinical Pharmacy Department, Faculty of Pharmacy, Tanta University, Tanta, Egypt (E.E.-K.); and Certara UK Ltd. (Simcyp Division), Sheffield, UK (A.R.-H.)
Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, UK (E.E.-K., Z.M.A.-M., A.R.-H., J.B., B.A.); Clinical Pharmacy Department, Faculty of Pharmacy, Tanta University, Tanta, Egypt (E.E.-K.); and Certara UK Ltd. (Simcyp Division), Sheffield, UK (A.R.-H.).
Drug Metab Dispos. 2021 Aug;49(8):610-618. doi: 10.1124/dmd.121.000484. Epub 2021 May 27.
Model-based assessment of the effects of liver disease on drug pharmacokinetics requires quantification of changes in enzymes and transporters responsible for drug metabolism and disposition. Different proteomic methods are currently used for protein quantification in tissues and in vitro systems, each with specific procedures and requirements. The outcome of quantitative proteomic assays using four different methods (one targeted and three label-free) applied to the same sample set was compared in this study. Three pooled cirrhotic liver microsomal samples corresponding to cirrhosis with nonalcoholic fatty liver disease, biliary disease, or cancer and a control microsomal pool were analyzed using quantification concatemer-based targeted proteomics, the total protein approach (TPA), high three ion intensity (Hi3) approach, and intensity-based absolute quantification (iBAQ) to determine the absolute and relative abundance in disease compared with control. The relative abundance data provided a "disease perturbation factor" (DPF) for each target protein. Absolute and relative abundances generated by standard-based label-free methods (iBAQ and Hi3) showed good agreement with targeted proteomics (limited bias and scatter), but TPA (standard-free method) overestimated absolute abundances by approximately 2-fold. The DPF was consistent between different proteomic methods but varied between enzymes and transporters, indicating discordance of effects of cirrhosis on various metabolism-related proteins. The DPF ranged from no change (e.g., for glucuronosyltransferase-1A6 in nonalcoholic fatty liver disease group) to less than 0.3 (e.g., carboxylesterases-1 in cirrhosis of biliary origin). SIGNIFICANCE STATEMENT: This study demonstrated that relative changes in enzymes and transporters (DPF) are independent of the quantitative proteomic methods used. Standard-based label-free methods, such as high three ion intensity (Hi3) and intensity-based absolute quantification (iBAQ) methods, were less biased and more precise than the total protein approach (TPA) when compared with targeted data. The DPF reconciled differences across proteomic methods observed with absolute levels. Using this approach, differences were revealed in the expression of enzymes/transporters in cirrhosis associated with different etiologies.
基于模型的肝疾病对药物药代动力学影响的评估需要定量检测负责药物代谢和处置的酶和转运体的变化。目前，有不同的蛋白质组学方法可用于组织和体外系统中的蛋白质定量，每种方法都有特定的程序和要求。本研究比较了四种不同方法（一种靶向和三种无标记）在同一样本集中进行定量蛋白质组学测定的结果。使用定量串联物靶向蛋白质组学、总蛋白法（TPA）、高三个离子强度（Hi3）法和基于强度的绝对定量（iBAQ），分析了三个对应于非酒精性脂肪性肝病、胆道疾病或癌症的肝硬化混合肝微粒体样本和一个对照微粒体池，以确定与对照相比疾病中的绝对和相对丰度。相对丰度数据为每个靶蛋白提供了“疾病扰动因子”（DPF）。基于标准的无标记方法（iBAQ 和 Hi3）生成的绝对和相对丰度与靶向蛋白质组学（偏差和分散有限）具有良好的一致性，但 TPA（无标准方法）高估了约 2 倍的绝对丰度。不同蛋白质组学方法之间的 DPF 是一致的，但在酶和转运体之间存在差异，表明肝硬化对各种代谢相关蛋白的影响不一致。DPF 范围从无变化（例如，非酒精性脂肪性肝病组中的葡萄糖醛酸转移酶-1A6）到小于 0.3（例如，胆道来源的肝硬化中的羧酸酯酶-1）。意义陈述：本研究表明，酶和转运体的相对变化（DPF）与使用的定量蛋白质组学方法无关。与靶向数据相比，基于标准的无标记方法（如 Hi3 和 iBAQ 方法）的偏差更小，更精确。DPF 协调了绝对水平观察到的蛋白质组学方法之间的差异。使用这种方法，揭示了不同病因引起的肝硬化中酶/转运体的表达差异。
